L4-6 DRGs (in control and sciatic nerve crush studies) were collected after euthanasia, total RNA was isolated by a PureLinkTM Micro-to-midi kit (Invitrogen), and cDNA was reverse-transcribed from mRNA with the Super-Script First Strand Synthesis System for reverse transcription-PCR (Invitrogen). Fluorescence-based quantitative real-time reverse transcription-PCR was carried out using the LightCycler 480 and with LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics). Primers were as follows: 5′-TGTATGCCTCTGGTCGTACC-3′ (sense) and 5′-CAGGTCCAGACGCAGGATG-3′ (antisense) for GAPDH; 5′-CTCAACTACATGGTCTACATGTTCCA-3′ (sense) and 5′- CCATTTCGGCCTTGACTGT-3′ (antisense) for AQP1, 5′-CAGGAAAGATCCCAAGTCCA-3′ (sense) and 5′-GAACGGAACATTGCACACAC-3′ (antisense) for GAP-43. Data were analyzed by LightCycler software 4.0 (Roche Diagnostics) and reported as calibrated ratios normalized to GAPDH. Data were averaged from six mice.
Purelink micro to midi kit
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The Purelink micro to midi kit is a nucleic acid purification system designed to extract and purify DNA and RNA from a variety of sample types. The kit utilizes spin column-based technology to facilitate efficient and reliable purification of nucleic acids.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (2 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (2 mentions)
Taqman probe, by Thermo Fisher Scientific (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Superscript first strand synthesis system for reverse transcription pcr, by Thermo Fisher Scientific (1 mentions)
Lightcycler software 4, by Roche (1 mentions)
Lightcycler faststart dna masterplus sybr green 1 kit, by Roche (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Bioarray rna labeling kit, by Enzo Life Sciences (1 mentions)
Superscript 2 reverse transcription, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
5 protocols using purelink micro to midi kit
1
Quantitative Analysis of Aquaporin-1 and GAP-43 Expression
2
Microarray Analysis of PKC Isoforms
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Gene expression studies were performed as described [33 (link)]. Briefly, RNA was extracted and purified with the PureLinkTM Micro-to-Midi kit (Invitrogen, Paisley, UK). cDNA and biotinylated cRNA were synthesized using a T7-polyT primer and the BioArray RNA labeling kit (Enzo, NY, USA), respectively. Labeled RNA was hybridized to Human Gene 1.0 ST oligonucleotide arrays (Affymetrix, CA, USA). For the microarray data analysis, the .CEL files (in triplicate) were imported into the dChip software 19, and expression levels of the different PKC isoforms analysed. Levels of expression (arbitrary units) of the different PKC isoforms were plotted.
3
Extracting RNA from Sciatic Nerves and Schwann Cells
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To extract total RNA from sciatic nerves and cultured rat Schwann cells, we used the Purelink Micro-To-Midi kit and followed the instructions of the manufacturer (Invitrogen). Genomic DNA was removed by incubation with RNase free DNase I (Fermentas), and RNA was primed with random hexamers and retrotranscribed to cDNA with Super Script II Reverse transcription (Invitrogen). Control reactions were performed omitting retrotranscriptase. qPCR was performed using the Applied Biosystems 7500 Real Time PCR System and 5× PyroTaq EvaGreen qPCR Mix Plus (CMB). To avoid genomic amplification, PCR primers were designed to fall into separate exons flanking a large intron when possible. Reactions were performed in duplicates of three different dilutions, and threshold cycle values were normalized to the housekeeping gene 18S. The specificity of the products was determined by melting curve analysis and gel electrophoresis. The ratio of the relative expression for each gene to 18S was calculated by using the 2ΔCT formula. Amplicons were of similar size (≈100 bp) and melting points (≈85°C). Similar amplification efficiency for each product was confirmed by using duplicates of three dilutions for each sample.
4
Quantitative RT-PCR for CPEB3 mRNA
5
Quantitative RT-PCR for CPEB3 mRNA
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Neurons were washed twice with ice-cold phosphate buffered saline (PBS) and scraped in TRIzol (Invitrogen) and RNA was extracted by means of the Purelink micro to midi kit (Invitrogen) or by phenol chloroform isolation. RNA concentration was measured by means of a Nanodrop (Nanodrop Technologies). Aliquots of RNA (1 μg) were reverse-transcribed to cDNA with SuperScript III Reverse Transcriptase (Invitrogen) using olido d(T) primers. Real-time qPCR was performed as described (Noh et al., 2012 (link); Rodenas-Ruano et al., 2012 (link)) with TaqMan probes (Applied Biosystems) for CPEB3 (reference number: Mm01204299_m1) and normalized to GAPDH (reference number: Mm99999915_g1). Reactions were performed in triplicate in a StepOnePlus real-time PCR system (Applied Biosystems). The relative change in mRNA expression was determined by the equation: Fold change = 2− ΔCt (ΔCt = Ct target – Ct reference) where Ct refers to cycle number at which the fluorescence signal crosses a threshold (Livak and Schmittgen, 2001 (link)). Relative expression ratio in Fmr1 KO neurons was calculated by normalization to WT type levels.
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